The Rho GTPases (Rac, Cdc42Hs and Rho) are implicated in the regulation of diverse biological responses including transcriptional activation, growth control and the reorganization of cell morphology. (Hall et al., Science, 1998, 279:509-514 and Van Aelst et al., Genes Dev., 1997, 11: 2295-2322) Microinjection of these GTPases into mammalian cells demonstrated that Cdc42Hs is implicated in the formation of filopodia (Nobes et al., Cell, 1995, 81: 53-62 and Kozma et al., Mol Cell Biol., 1995, 15: 1942-1952) and activation of Rac to induce lamellipodia (Ridley et al., Cell, 1992, 70: 401-410) and subsequently activates Rho to induce stress fibers (Ridley et al., Cell, 1992, 70: 401-410). Over the last few years, various down stream molecular targets have been identified for these GTPases. These targets interact with the activated GTP bound forms of the GTPases. Cdc42Hs and Rac have been shown to interact specifically with WASP (Aspenstrom et al., Curr Biol., 1996, 6: 70-75; Symons et al., Cell, 1996, 84: 723-734; and Kolluri et al., Proc Natl Acad Sci., 1996, 93: 6515-5618), IQGAP (Hart et al., EMBO J., 1996, 15: 2997-3005; and Kurodas et al., J. Bio. Chem., 1996, 271: 23363-23367), POSH (Tapon et al., EMBO J., 1998, 17: 1395-1404), POR1 (Van Aelst et al., EMBO J 1996, 115: 3778-3786) p67-phox (Diekmann et al., Science,1994, 265: 531-533), MLK3 (Teramoto et al, J Biol Chem., 1996, 271: 27225-27228) and PAKs (Maser et al., Nature, 1993, 363: 364-357; Martin et al., EMBO J 1995, 14: 1997-1978; and Bagrodia et al., J Biol Chem., 1995, 270: 27995-27998). The p21 activated kinase (PAK) is one of the downstream effectors for the GTPases Rae and Cdc42Hs (Maser et al., Nature 1993, 363: 364-357; Martin et al., EMBO J 1995, 14: 1997-1978; Bagrodia et al., J Biol Chem 1995, 270: 27995-27998; and Sells et al., Trends Cell Biol 1997, 7: 1623-167). The GTP bound form of the GTPases interacts with PAK through a conserved GTPase binding domain (GBD) and subsequently stimulates Pak activity (Maser et al., Nature 1993, 363: 364-357; Martin et al., EMBO J 1995, 14: 1997-1978; Bagrodia et al., J Biol Chem 1995, 270: 27995-27998; and Sells et al., Trends Cell Biol 1997, 7: 1623-167). Three mammalian PAKs have been identified including PAK1, 2 mouse PAK3 and three rat homologues PAK, alpha, beta, and gamma (Sells et al., Trends Cell Biol 1997, 7: 1623-167).
All of the PAKs contain a regulatory domain (PakR) and a highly conserved kinase domain that is closely related to yeast STE20 (Sells et al., Trends Cell Biol 1997, 7: 1623-167). The PakR contains the GBD and a polyproline sequence important for interaction with SH3 domain containing proteins (Bagrodia et al., J Biol Chem 1995, 270: 27995-27998. A possible role for PAK function came from studies of the yeast homologue STE20, a protein kinase that is implicated in the activation of the MAP kinase cascade in Saccharomyces cerevisiae. Several groups have shown that mammalian PAK can activate the JNK MAP kinase cascade (Minden et al., Cell 1995, 81: 147-1157 and Coso et al., Cell 1995, 81: 1137-1146) and recent work with effector mutants of Rac and Cdc42Hs suggests that PAK is necessary for JNK activation (Lamarche et al., Cell 1996, 87: 519-529). The fact that PAKs are effectors for Rae and Cdc42Hs suggested that they may also play a role in the regulation of the reorganization of the actin cytoskeleton. Several reports have demonstrated the recruitment of PAK to the focal complexes and lamellipodia, however, these effects are independent of PAK kinase activity and interaction with Cdc42Hs or Rae (Dharmawardhane et al., J Cell Biol 1997, 138: 1265-1278; Sells et al., Curr Biol 1997, 7: 202-210 and Manser et al., Mol Cell Biol 1997, 17: 1129-1143). Moreover, effector mutants of Rac and Cdc42Hs that fail to interact with PAKs are able to promote the induction of lamellipodia and filopodia (Lamarche et al., Cell 1996, 87: 519-529).
Pak2 interacts with the SH3 domains of NCK (Bagrodia et al., J Biol Chem 1995, 270: 27995-27998) and Grb2 through its proline rich sequence. Recently, Manser et al., Mol Cell 1998, 1: 183-192 demonstrated that PakR contains a unique proline sequence that can bind an SH3 domain of a novel GTP/GDP exchange factors-PIX. It is suggested that PIX/PAK interaction is important for the association of PAK with the focal complexes (Manser et al., Mol Cell 1998, 1: 183-192).